Edward P. Whitehead

A reverse-phase HPLC method for cAMP phosphodiesterase activity

A simple and fast method based on reverse-phase HPLC has been developed for measuring the activity of cAMP phosphodiesterase. It allows quantitation of product and substrate in less than 10 min. The sensitivity (1*10(-11) mol AMP), the accurate evaluation of nucleotides, the unequivocal analysis of product, and the reproducibility of the system, make this method suitable for the evaluation of cAMP phosphodiesterase in biological material, at different levels of purification, and also in kinetic studies.

Allosteric modifier and substrate binding of donkey deoxycytidylate aminohydrolase (EC 3.5.4.12)

The hexameric allosteric enzyme deoxycytidylate aminohydrolase from donkey spleen is shown by equilibrium dialysis to bind specifically the allosteric inhibitor, dTTP, the activator dCTP, and the substrate analog dAMP each at six sites (the dTTP and dCTP sites may or may not be identical). These conclusions contrast with earlier ones that there were four sites for each effector; reasons for the discrepancy are discussed. With the knowledge of site numbers and the kinetic information from the accompanying paper it is concluded that the kinetic cooperativity of the enzyme excludes a concerted conformational transition mechanism. Amino acid analysis gives a molecular weight of 18,842 Da per subunit, i.e., 113,052 for the hexamer. A new simplified purification of homogeneous enzyme from donkey spleen probably useful for dCMP aminohydrolase from other sources is described.

Hill coefficient ratios give binding ratios of allosteric enzyme effectors; inhibition, activation, and squatting in deoxycytidylate aminohydrolase (EC 3.5.4.12)

The ratio of the steady-state kinetic Hill coefficients of two different effectors equals (under some rather weak general assumptions) the ratio in which the effectors displace each other from an enzyme. This principle can make implications of experimental allosteric enzyme kinetic data immediately apparent. We can use it to find that one molecule of the allosteric inhibitor of dCMP aminohydrolase, at moderately high effector concentrations, displaces one molecule of substrate, or one molecule of activator, whereas at very high concentrations, one molecule of inhibitor displaces two of substrate. Further use of the principle suggests that substrate, at high concentrations, binds binds to activator sites. However, ratios of substrate, activator, and inhibitor Hill coefficients are incompatible with a simple model of activation in which substrate and activator are bound to the same conformation.

ATP-dependent exonuclease V from Micrococcus luteus The enzyme-DNA complex, the processive mechanism, and the role of ATP

Some kinetic predictions of the proposed processive mechanism for the hydrolysis of DNA by the ATP-dependent enzyme exonuclease V have been checked. The method is to trap enzyme molecules not attached to radioactive DNA substrate with an excess of nonradioactive DNA, so that enzyme molecules attached to the radioactive substrate contribute to the liberation of radioactive products only until they dissociate from it. The experiments show that enzyme molecules remain attached to a T7 double-stranded DNA molecule, while hydrolysing it, for about 2 min under our conditions, in agreement with the predictions of the processive mechanism. However, the mechanism of degradation of single-stranded DNA is not processive. Formation of an enzyme-DNA complex is largely dependent on the presence of ATP. This formation does not appear to be synchronous. ATP analogs do not stimulate formation of, nor stabilize, the enzyme-DNA complex. EDTA causes dissociation of enzyme molecules from the DNA complex.

Measurement of DNA methylase activity by tritium release from DNA cytosine

The advantages of assaying of DNA methylase by measuring the transfer to water of tritium from the 5 position of DNA cytosine, rather than the transfer to DNA of labeled methyl groups are discussed.